In-vitro model of the Penumbra: closed-loop optogenetic stimulation to improve cell survival.
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1
University of Twente, Clinical Neurophysiology, Netherlands
Introduction
Stroke is one of the major causes of impairments or death. Impeded blood supply in the infarct core causes loss of neuronal function, followed by neuronal death within minutes. In peripheral areas of an infarct, the penumbra, neurons are functionally silent, but structurally intact and viable due to some remaining perfusion from surrounding arteries. Here, dysfunction is in principle reversible, which makes the penumbra a promising target for treatment. However, there are no therapies to promote recovery of penumbral tissue, at least in part because details on the mechanisms are unclear. The observation that the total amount of activity generally decreased, and the notion that neurons must be active to survive, suggest that hypoxia induced apoptosis is possibly triggered by insufficient neuronal activity. In this study, we used neuronal network cultured on micro electrodes arrays (MEAs) combined with optogenetic tools, to monitor the neuronal viability while controlling the extracellular condition and cellular activities.
Methods
Cells were obtained from brain cortices of newborn Wistar rats on the day of the birth and plated on a micro electrode array (MEA) precoated with poly ethylene imine (PEI). MEAs were placed under a Plexiglas hood, and a humidified gas mixture of air and N2 supplemented with 5% CO2, was blown over the setup at a rate of 2 l/min. Mixtures of air and N2 could be delivered at any ratio and were computer controlled by mass flow controllers. Normoxic conditions were realized by setting the flow controllers to 100% air; hypoxic mixtures contained 10% air and 90% N2.
To enable light stimulation, we used an Adeno Associated Virus serotype 1 (AAV1) containing the ChannelRhodopsin-2 gene driven by the neurons CaMKIIα promotor. This opsin is a trans membrane protein that conducts cations and depolarizes neurons upon blue light (λ=470nm) illumination, while the promoter selectively restricts expression of virally delivered genes to excitatory neurons. To assess the characteristics of the response to light stimulation in ‘tuning curves’, a randomized stimulation protocol was implemented. Different stimuli of varying duration (10-1000ms) and intensity (2.5-11 klx) were delivered in a randomized order (n=30 per stimulus) every 10s, and electrical activity was recorded to determine the optimum stimulus parameters. To obtain tuning curves, responses to stimuli with varying duration or intensity were quantified by the area under the stimulus-response curves (with latencies ≤1000ms). Then, curves were normalized to their maximum value. To determine whether cells were also able to respond to light stimulation under hypoxic conditions, a series of light pulses was delivered after 1h, 2h or 5h of hypoxia (with no earlier stimulation).
Experiments consisted of six hours baseline under normoxia, 24 hours of hypoxia and another six hours after reoxygenation. For the quantification of the amount of activity during experiments, we determined the array-wide firing rate (AWFR) as the summed number of action potentials of all electrodes in 1h time bins. Cultures were light stimulated during the hypoxic phase if the activity dropped below a threshold (set at 45% of baseline activity), using two different regimes of closed loop stimulation: Duration Loop and Intensity Loop. Duration Loop - During hypoxia, the number of stimuli (fixed at 9.2klx) per min was incremented (starting from 0 pulses (10ms) per minute), whenever the recorded activity in the previous minute was below threshold, and decremented if the activity in the previous minute was above threshold. Stimuli were evenly distributed, with a maximum of twelve per minute. When this maximum was reached, the pulse duration was increased (with a maximum at 1s) and the number of pulses was reset to 1 stim/min.
Intensity Loop - The other regime is equal to the first, but with increasing light intensity (2.5-11klx) instead of increasing duration (which was fixed at 200ms). When the stimulus reached maximum intensity, the number of stimulation per mins was reset to 1 and did not increase anymore.
Recovery after hypoxia was assessed by the AWFR and the number of active electrodes (NAE) in the first six hours after reoxygenation, both normalized to their baseline values. In control experiments, cultures were transfected, but not light stimulated during hypoxia.
Results
Stimulus-response curves obtained from different cultures differed in shape, even when duration and intensity were equal (Fig 1A). Tuning curves, however, were comparable across cultures, showing that cultures were more sensitive to increasing pulse duration than to increasing intensity (Fig 1B,C). Also, during hypoxia, clear responses to light stimulation could still be recorded, but their magnitude decreased with longer hypoxia.
In all experiments activity eventually dropped below the set threshold of 45% of baseline, despite maximum stimulation. Under the Intensity Loop regime, cultures were most active during hypoxia and showed significantly higher AWFR (t-test: p<0.03) after reoxygenation than Controls (n=5). Cultures under Duration Loop (n=4) showed similar recovery as Controls (p=0.3).
2-7 days after the experiments, all cultures were checked for activity. Whereas none of the Controls and Duration Loop cultures showed any activity, four of the six Intensity Loop cultures had remained active.
Discussion
We showed that light stimulation was possible, even under severe hypoxic conditions. Despite the inability to maintain activity around 45% of baseline, the Intensity Loop yielded significantly better recovery than Control or the Duration Loop. The main difference between both closed loop regimes was that increasing duration was received as a stronger stimulus, and increasing intensity was not. Moreover, in the Intensity Loop the stimulation frequency was limited to 1 pulse/min when maximum intensity was reached, compared to 12 pulses/min of maximum duration in the duration loop protocol. Possibly, neurons were overstimulated in the duration loop experiments, which may be harmful under hypoxia induced ATP scarcity. The Intensity Loop experiments show that it was possible to significantly improve recovery using light stimulation during severe hypoxia. Still, we did not take full advantage of the closed loop protocol. Parameter settings may not have reached the optimum, and future experiments will be directed at determination of the optimum balance between activity and available energy (ATP).
Keywords:
Ischemic Penumbra,
Recovery of Function,
Hypoxia, Brain,
activity dependent cell survival,
photo stimulation
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Oral Presentation
Topic:
Stimulation strategies
Citation:
Muzzi
L,
Hassink
GC and
Le Feber
J
(2019). In-vitro model of the Penumbra: closed-loop optogenetic stimulation to improve cell survival..
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00046
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Received:
15 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Dr. Joost Le Feber, University of Twente, Clinical Neurophysiology, Enschede, 7500AE, Netherlands, j.lefeber@utwente.nl